A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants

The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.     

1-(1-acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea (AR9281) as a potent, selective, and orally available soluble epoxide hydrolase inhibitor with efficacy in rodent models of hypertension and dysglycemia

1-(1-Acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea 14a (AR9281), a potent and selective soluble epoxide hydrolase inhibitor, was recently tested in a phase 2a clinical setting for its effectiveness in reducing blood pressure and improving insulin resistance in pre-diabetic patients. In a mouse model of diet induced obesity, AR9281 attenuated the enhanced glucose excursion following an intraperitoneal glucose tolerance test. AR9281 also attenuated the increase in blood pressure in angiotensin-II-induced hypertension in rats. These effects were dose-dependent and well correlated with inhibition of the sEH activity in whole blood, consistent with a role of sEH in the observed pharmacology in rodents.     

Au-nanocluster emission based glucose sensing

Fabrication of a glucose biosensor based on Au-cluster emission quenching in the UV region is reported. The glucose biosensor is highly sensitive to β-d-glucose in 2.5-25.0mM range as confirmed from a linear calibration plot between Au-cluster colloid emission intensity as a function of β-d-glucose concentration. The interaction of β-d-glucose with l-cysteine capped Au cluster colloids has been confirmed from their Fourier transformed infrared spectroscopy (FTIR) measurements. It has been found that the biomolecules present in the serum such as ascorbic and uric acids, proteins and peptides do not interfere and affect in glucose estimation as confirmed from their absorption and fluorescence (FL) emission measurements. Practical utility of this sensor based on FL quenching method has been demonstrated by estimating the glucose level in human serum that includes diabetes and the data were found to be comparable or more accurate than those of the pathological data obtained from a local hospital. In addition, this biosensor is useful to detect glucose level over a wide range with sensor response time of the order of nano to picoseconds that is emission lifetime of Au clusters.     

Evaluation of lead and chromium tolerance and accumulation level in Gomphrena celosoides: a novel metal accumulator from lead acid battery waste contaminated site in Nigeria

Abstract

Biology, tolerance, and metal (Pb and Cr) accumulating ability of Gomphrena celosoides were studied under hydroponic conditions. The seedlings were raised in Hoagland’s solution containing different concentrations of Pb (0, 500, 1000, 1500, 2000, 3000, 4000, and 5000?mg l^?1) and Cr (0, 50, 100, 150, 200, 300, and 400?mg l^?1). Biomass and metal accumulation in different plant parts were determined at seven (7) and fourteen (14) days after stress. Antioxidant enzyme activities, protein, and proline contents were estimated in stressed and unstressed plants. Gomphrena celosoides was able to tolerate Pb and Cr concentrations up to 4000 and 100?mg l^?1, respectively in hydroponic solution. Metal accumulation was concentration and duration dependent with the highest Pb (21,127.90 and 117,985.29?mg kg^?1) and Cr (3130.85 and 2428.90?mg kg^?1) in shoot and root, respectively found in the plants exposed to 5000?mg l^?1 Pb and 400?mg l^?1 Cr for 14?days. Proline, antioxidant enzyme activities, and protein contents were the highest in plant exposed to higher Pb and Cr concentrations for 7 and 14?days. Gomphrena celosoides could be considered as Pb and Cr accumulator with proline and increase in antioxidant enzyme activities being the tolerance mechanisms.     

Common Myna Roosts Are Not Recruitment Centres

We studied communal roosting in the Common Myna (Acridotheres tristis) in the light of the recruitment centre hypothesis and predation at the roost. The number and sizes of flocks departing from and arriving at focal roosts were recorded over a two year period. We also recorded the sizes and behaviour of foraging flocks. We found that flock sizes of birds departing from roosts at sunrise were larger than those at the feeding site, suggesting that there was no recruitment from the roosts. Flocks entering the roosts during sunset were larger on average than those leaving the following sunrise, suggesting no consolidation of flocks in the morning. Flocks entering the roosts at sunset were also larger on average than those that had left that sunrise, although there was no recruitment at the feeding site. There was no effect of group size on the proportion of time spent feeding. Contrary to expectation, single birds showed lower apparent vigilance than birds that foraged in pairs or groups, possibly due to scrounging tactics being used in the presence of feeding companions. Thus, the recruitment centre hypothesis did not hold in our study population of mynas. Predation at dawn and dusk were also not important to communal roosting: predators near the roosts did not result in larger flocks, and resulted in larger durations of arrival/departure contrary to expectation. Since flock sizes were smallest at the feeding site and larger in the evening than in the morning, but did not coincide with predator activity, information transfer unrelated to food (such as breeding opportunities) may possibly give rise to the evening aggregations.     

Methane production in rice soil is inhibited by cyanobacteria

The present study was aimed at understanding the role of cyanobacteria and Azolla in methane production and oxidation in laboratory simulation experiments using soil samples from rice fields. All the seven cyanobacterial strains tested effected a significant decrease in the headspace concentration of methane in flooded soil, incubated under light. Synechocystis sp. was the most effective in retarding methane concentration by 10-20 fold over that in controls without cyanobacteria. The decrease in the headspace concentration of methane was negligible in nonsterile soil samples, inoculated with Synechocystis sp. and then incubated under dark. Moist soil cores (0-5 cm depth), collected from rice fields that had been treated with urea in combination with a cyanobacterial mixture, Azolla microphylla, or cyanobacterial mixture plus A. microphylla, effected distinctly more rapid decrease in the headspace concentration of methane added at 200 microl(-1) than did the soil cores from plots treated with urea alone (30, 60, 90 and 120 kg N ha(-1)), irrespective of the rate of chemical nitrogen applied to rice fields. Besides, soil cores from plots treated with urea alone at 60, 90 and 120 kg N ha(-1) oxidised methane more rapidly than did the core samples from plots treated with urea alone at 30kg N ha(-1). Cyanobacteria and A. microphylla, applied to flood water, appear to play a major role in mitigation of methane emission from rice fields-through enhanced methane oxidation.     

Sirt1 and mir-9 expression is regulated during glucose-stimulated insulin secretion in pancreatic β-islets

MicroRNA mir-9 is speculated to be involved in insulin secretion because of its ability to regulate exocytosis. Sirt1 is an NAD-dependent protein deacetylase and a critical factor in the modulation of cellular responses to altered metabolic flux. It has also been shown recently to control insulin secretion from pancreatic β-islets. However, little is known about the regulation of Sirt1 and mir-9 levels in pancreatic β-cells, particularly during glucose-dependent insulin secretion. In this article, we report that mir-9 and Sirt1 protein levels are actively regulated in vivo in β-islets during glucose-dependent insulin secretion. Our data also demonstrates that mir-9 targets and regulates Sirt1 expression in insulin-secreting cells. This targeting is relevant in pancreatic β-islets, where we show a reduction in Sirt1 protein levels when mir-9 expression is high during glucose-dependent insulin secretion. This functional interplay between insulin secretion, mir-9 and Sirt1 expression could be relevant in diabetes. It also highlights the crosstalk between an NAD-dependent protein deacetylase and microRNA in pancreatic β-cells.     

Evaluation of vitamin D3 A-ring analogues as Hedgehog pathway inhibitors

A structure-activity relationship study focusing on the A-ring of vitamin D3 (VD3) was undertaken to elucidate its role in inhibiting the Hedgehog pathway and in mediating anti-cancer effects. Analogues resulting from simple functional group substitution at 3' position of VD3 were evaluated in a variety of biological assays to determine their ability to selectively inhibit Hh signaling. Moderately active Hh inhibitors that have insignificant binding affinity for VDR were identified; however, these compounds also activate the traditional VDR pathway, presumably due to metabolites produced in the cultured cells. Thus, further structural modifications to the VD3 scaffold are required to yield potent, selective Hh inhibitors.     

An AICD-based functional screen to identify APP metabolism regulators

Background

The generation of the amyloid-β peptide (Aβ) through the proteolytic processing of the amyloid precursor protein (APP) is a central event in the pathogenesis of Alzheimer's disease (AD). Recent studies highlight APP endocytosis and localization to lipid rafts as important events favoring amyloidogenic processing. However, the precise mechanisms underlying these events are poorly understood. ApoER2 is a member of the low density lipoprotein receptor (LDL-R) family exhibiting slow endocytosis rate and a significant association with lipid rafts. Despite the important neurophysiological roles described for ApoER2, little is known regarding how ApoER2 regulates APP trafficking and processing.

Results

Here, we demonstrate that ApoER2 physically interacts and co-localizes with APP. Remarkably, we found that ApoER2 increases cell surface APP levels and APP association with lipid rafts. The increase of cell surface APP requires the presence of ApoER2 cytoplasmic domain and is a result of decreased APP internalization rate. Unexpectedly, ApoER2 expression correlated with a significant increase in Aβ production and reduced levels of APP-CTFs. The increased Aβ production was dependent on the integrity of the NPxY endocytosis motif of ApoER2. We also found that expression of ApoER2 increased APP association with lipid rafts and increased γ-secretase activity, both of which might contribute to increased Aβ production.

Conclusion

These findings show that ApoER2 negatively affects APP internalization. However, ApoER2 expression stimulates Aβ production by shifting the proportion of APP from the non-rafts to the raft membrane domains, thereby promoting β-secretase and γ-secretase mediated amyloidogenic processing and also by incrementing the activity of γ-secretase.